Blockers of the growth hormone receptor in disease prevention and treatment

ABSTRACT

Compounds and methods for treating diseases or conditions affected by the activity or expression of genes/proteins related to human GH, GHR, STAT5, SOCS, IGF-1, insulin are provided. Monoclonal antibodies for treating diseases or conditions related to growth hormone and growth hormone receptor activity are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 62/115,356 filed Feb. 12, 2015, the disclosure of which is herebyincorporated in its entirety by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The invention was made with Government support under Contract No.

-   -   5P01AGO34906-04 awarded by the National Institute of Health. The        Government has certain rights to the invention.

SEQUENCE LISTING

The text file is usc0138_ST25.txt, created Feb. 12, 2016, and of size 8KB, filed herewith, is hereby incorporated by reference.

TECHNICAL FIELD

In at least one aspect, the present invention relates to compounds andmethods for treating diseases or conditions by causing inhibition of theactivity or expression of GH, GHR, STAT5, IGF-1 and/or SOCS and ofproteins regulated by human GH, GHR, STAT5, SOCS, IGF-1, and insulin.

BACKGROUND

Acromegaly is a disease caused by excessive secretion of GH frompituitary adenomas. The estimated world-wide market for SOMAVERT®(pegvisomant a human GHR antagonist) is over 160 M USD. SOMAVERT® isadministered to patients who have failed pituitary adenoma surgery andare resistant to somatostatin analogs [1] A major downside to SOMAVERT®treatment is that it is administered as a once daily injection (Pfizer).Thus a GHR blocker such as that being proposed here, that can block hGHRsignaling would be very desirable in the treatment of acromegaly.

Age is a major risk factor for many types of tumors resulting in amarkedly increased cancer incidence in the elderly population [2-4].Most cancers (78%) are diagnosed in persons 55 years of age and older(source: American Cancer Society). Age is also associated with increasedchemotherapy toxicity, which limits the safety and efficacy of standardchemotherapy [5-7]. In clinical reports, elderly patients experiencedmore myelosuppression and had a greater risk of chemotherapy-relateddeath than younger patients in many types of cancers [6]. This poses aserious problem considering that most cancers occur in elderlyindividuals who are also more susceptible to chemotherapy toxicity.Although new and less toxic drugs are slowly replacing or being added tothe widely used toxic chemotherapy drugs, interventions to reducetoxicity in the elderly are not established [8]. As underlined recentlyin Nature Reviews in Clinical Oncology in an article tided “Reducing thetoxicity of cancer therapy: recognizing needs, taking action”development of novel strategies and drugs aimed at selectivehost/patient protection could reduce the side effects associated withchemotherapy treatment and also increase the therapeutic index. Becausethese drugs would protect against both exogenous and endogenous toxins,they would also have the potential to protect against age-related damageand diseases including cancer, diabetes and neurodegenerative diseases.

It is estimated that by 2030 roughly 20% of the American population willbe comprised of individuals 65 and older (source cdc.gov). Chronicdiseases such as heart disease, cancer, Alzheimer's disease and diabetesare the most frequent causes of mortality in the elderly. About 95% ofhealth care costs among older adults (65 and older) go towards chronicdiseases and these costs are expected to increase by 25% by 2030(CDC.gov). Thus, there is a great emphasis on preventing and/or delayingchronic health conditions in the aging population not only to improvequality of life in the elderly but also to curtail rising health carecosts.

The basis for targeting the growth hormone receptor (GHR) is as follows.Mutations that cause genetic inhibition of the GH/GHR/IGF-1 lead to asmuch as a 50% increase in life span in mice [9-11]. Homozygous Amesdwarf mutations in the Prop-1 gene (df/df) prevent the generation of theanterior pituitary cells that produce growth hormone, thyroidstimulating hormone, and prolactin. Young adult df/df mice areapproximately one third of the size of control mice but survive >50%longer [9]. This effect of dwarf mutations on life span appears to becaused by the absence of plasma GH, which stimulates the secretion ofIGF-1 from liver cells [12]. In fact, IGF-1 is reduced dramatically inthe plasma of df/df mice. The plasma GH deficiency appears to mediatethe effects of Prop-1 (Ames dwarf) and Pit-1 (Snell dwarf) mutations onlongevity, since the mice that cannot release GH in response to growthhormone releasing hormone (GHRH) also live longer [12]. Furthermore,dwarf mice with high plasma GH, but a 90% lower circulating IGF-1(growth hormone receptor/GH binding protein knock mice, GHRKO) livelonger than their wild type littermates [10]. Taken together thesestudies suggest that the reduction in plasma IGF-1 and probably insulinis responsible for a significant portion of the life span increase indwarf, GH deficient, and GHR/BP null mice. In fact, mice lacking onecopy of IGF-1 receptor (IGF-IR^(+/−)) live 33% longer than their wildtype controls [11]. As observed in long-lived lower eukaryotes, theactivities of antioxidant enzymes superoxide dismutases and catalase aredecreased in murine hepatocytes exposed to GH or IGF-1 and in transgenicmice overexpressing GH [13, 14]. In vitro studies with fibroblasts frommutant mice with deficiencies in the GH/IGF-1 axis show increasedresistance against various types of stress including UV, H₂O₂, paraquat,alkylating agent, heat and cadmium [15]. In rats, IGF-1 attenuatescellular stress response and the expression of stress response proteinsHSP72 and hemeoxygenase [16]. Studies by Longo and colleagues in primaryneurons suggest that IGF-1 sensitizes cells to oxidative stress by aRas/Erk-dependent mechanisms [17]. The Longo laboratory and others havedescribed how mutations that decrease the activity of the Tor/Sch9(homolog to mammalian AKT and S6K) pathway or of the adenylylcyclase/cAMP/PKA pathway increase life span and stress resistance inyeast [18-20]. The increase in resistance to oxidants and heat, forexample, can reach 1,000 fold in yeast with mutations in both pathways[21].

Recently, a reduction in adenylyl cyclase activity by deletion of theadenylyl cyclase 5 (AC5) gene was also shown to extend life span andincrease resistance to oxidative stress in mice [22], suggesting thatpathways including homologs of Akt, S6 kinase and cAMP/PKA may play apartially conserved role in the regulation of aging and stressresistance in organisms ranging from yeast to mice (FIG. 2) [23].Analogous to the activation of yeast Sch9 and Ras by glucose, themammalian IGF-1 receptor activates both Akt/mTOR/S6K and Ras, andregulates glucose metabolism and cellular proliferation [24].Accumulating evidence has implicated increased IGF-1 or IGF-1 signalingas risk factors in a variety of cancers [25], suggesting that thispro-mitotic pathway can promote aging and also the damages and mutationsnecessary for tumorigenesis.

More recently a study of 99 living and 53 deceased Ecuadorianindividuals with genetic inhibition of the GHR (Growth Hormone ReceptorDeficient, GHRD) has shown that absence of GH/IGF-1 signaling protectsagainst two major age-related diseases, cancer and diabetes [26]. GHRDindividuals, who have very low IGF-1 levels appear to have a normal lifespan that is similar to their non-GHRD counterparts [26]. Thusinhibition of GH/IGF-1 signaling by drug interventions has the potentialto be useful in reducing the incidence of cancer and diabetesparticularly in families with a high incidence of these diseases. Asdiscussed earlier, inhibition of GH/IGF-1 signaling would have manyother applications: chronic treatment of acromegaly (excessive GHproduction) [27], differential protection against chemotoxicity [28],and oxidative stress associated with ischemia/reperfusion-induceddamages [29].

The inhibition of GHR-IGF-1 signaling has also recently been shown topromote hematopoietic regeneration (Cheng et al., Cell Stem Cell 2014)and stem cell based regeneration of multiple systems (U.S. Pat. Appl.No. 20140227373). Moreover, the genetic inhibition of the GHR protectsmice from chemotherapy-induced immune suppression (FIG. 3A) and DNAdamage in bone marrow and mononuclear peripheral blood cells, in part bycausing hematopoietic stem-cell dependent regeneration (FIG. 3B) (Chenget al., Cell Stem Cell 2014). It has also been demonstrated that thegenetic inhibition of the GHR reduces tumor growth and enhances thesurvival in a xenograft tumor model in mice (FIG. 4). Finally, Parrellaet al. have recently shown that inhibition of GH-IGF-1 signalingprotects from the age-dependent cognitive impairment and pathology in anAlzheimer's disease mouse model (Parrella et al., Aging Cell. 2013April; 12(2):257-68).

Accordingly, there is a need for developing new disease treatmentprotocols based on the inhibition of GHR-IGF-1 signaling.

SUMMARY

The present invention solves one or more problems of the prior art byproviding in at least one embodiment, a compound for treating diseasesor conditions by causing inhibition of the activity or expression of GH,GHR, STAT5, IGF-1 and/or SOCS and of proteins regulated by human GH,GHR, STAT5, SOCS, IGF-1, and insulin is provided. The compound of thisembodiment has formula I:

wherein:R₁ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen, andR₂ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen.

In another embodiment, a compound that is useful for the treatment ofdiseases or conditions by causing inhibition of the activity orexpression of GH, GHR, STAT5, IGF-1 and/or SOCS and of proteinsregulated by human GH, GHR, STAT5, SOCS, IGF-1, and insulin is provided.The compound of this embodiment has formula II:

wherein:R₁ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen, andR₂ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen.

In another embodiment, a compound that is useful for the treatment ofdiseases or conditions by causing inhibition of the activity orexpression of GH, GHR, STAT5, IGF-1 and/or SOCS and of proteinsregulated by human GH, GHR, STAT5, SOCS, IGF-1, and insulin is provided.The compound of this embodiment has formula III:

wherein:R₁ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen, andR₂ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen.

In another embodiment, a compound that is useful for the treatment ofdiseases or conditions by causing inhibition of the activity orexpression of GH, GHR, STAT5, IGF-1 and/or SOCS and of proteinsregulated by human GH, GHR, STAT5, SOCS, IGF-1, and insulin is provided.The compound of this embodiment has formula IV:

wherein:R₁ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen, andR₂ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen.

Advantageously, the compounds set forth above in formulae I-IV areuseful for treating diseases or conditions selected from the groupconsisting of acromegaly, cancer, diabetes, Alzheimer's, and aging. Thecompounds and inhibition of the pathways by the compounds are alsouseful to protect from chemotherapy toxicity and promote multi-systemstem cell based regeneration and to treat conditions and diseasesbenefiting from stem cell-based regeneration (C. W. Cheng et al. CellStem Cell, 2014; 14 (6): 810 DOI: 10.1016/j.stem.2014.04.014)

In another embodiment, a method for treating diseases related to growthhormone activity is provided. In this embodiment, inhibitory anti-growthhormone receptor (GHR) monoclonal antibodies are used to reduce growthhormone (GH) action and consequently the Insulin Growth Factor 1 (IGF-1)activity in vii. Because of the pro-growth effect of GH and IGF-1 oncertain tumor cells and because GH/IGF-1 signaling plays an importantrole in mammalian aging, this invention has a great potential to providean antibody-based drug to: a) attenuate/delay the growth of GH- and/orIGF-1 dependent tumor cells, b) induce organism-level protection againstacute stress, e.g. chemotherapy-associated toxicity to normal tissue,radiation induced cellular toxicity, or other toxic drugs and compounds;c) enhance the therapeutic index of existing chemotherapy; d) modulaterisk factors associated with age-related diseases; e) attenuate/delaychanges in biomarkers associated with aging; f) promote multi-systemstem cell-based regeneration; g) reduce or delay the incidence ofdiabetes and Alzheimer and retard their progression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Bar chart showing causes of death in the elderly (from the CDC,National Center for Health Statistics, National Vital Statistics System,2007).

FIG. 2. Similar pathways regulate longevity and resistance to stress inyeast and mice (adapted from Longo, 2003).

FIG. 3A. Genetic inhibition of the GHR protects bone marrow (BM) andmononuclear peripheral blood (PR) cells against chemotoxicity in mice.Total white blood cell (WBC) and lymphocyte counts in the peripheralblood of GHRD mice and their wild type littermates (WT); each pointrepresents the mean±s.e.m; vertical dashed lines indicatecyclophosphamide (CP) treatments; horizontal dashed lines indicatebaseline levels; * p<0.05, Two-way ANOVA for recovery phases.

FIG. 3B. GHR knockout (GHRKO) mice and their age-matched wild type (WT)littermates were treated with six cycles of cyclophosphamide (CP) (200mg/kg, i.p.). DNA damage (olive tail moment) were measured with CometAssay in bone marrow (BM) and mononuclear peripheral blood (PB) cellsafter 6 cycles of CP treatments.

FIG. 4A. Genetic inhibition of GHR reduces tumor growth and enhancestumor-bearing survival in a xenograft tumor model in mice. GHRKO miceand their age-matched wide type littermates (WT), were inoculated withB16Flu melanoma subcutaneously and subjected to Cyclophosphamide (CP)treatment (i.p. 200 mg/kg body weight, indicated with vertical dashedlines). Tumor growth over 3 cycles of CP treatment.

FIG. 4B. Tumor-bearing survival of the GHRKO and WT mice. Geneticinhibition of GHR reduces tumor growth and enhances tumor-bearingsurvival in a xenograft tumor model in mice. GHRKO mice and theirage-matched wide type littermates (WT), were inoculated with B16Flumelanoma subcutaneously and subjected to cyclophosphamide (CP) treatment(i.p. 200 mg/kg body weight, indicated with vertical dashed lines).

FIG. 5A. Lead compounds (#34, 37, 38) identified by luciferase reporterassays. Mouse L cells expressing human GHR were transfected with c-fosor SOCS2 reporter luciferase plasmids. Cells were serum starved,incubated with test compounds (#34, 37, 38) and then treated with GH.Data are normalized to co-transfected CMV-driven renilla luciferase andexpressed as relative light units (RLU). (A) c-fos reporter luciferaseactivity.

FIG. 5B. Lead compounds (#34, 37, 38) identified by luciferase reporterassays. Mouse L cells expressing human GHR were transfected with c-fosor SOCS2 reporter luciferase plasmids. Cells were serum starved, thenincubated with test compounds (#34, 37, 38) before GH treatment. Datawere normalized to co-transfected CMV-driven renilla luciferase andexpressed as relative light units (RLU). (B) SOCS2 reporter luciferaseactivity.

FIG. 6. Lead compounds inhibit STAT5 phosphorylation. (A) Mouse L cellswere incubated in 0.5% serum for 24 hours. Cells were treated withcompounds 34, 37 or 38 for 30 min prior to 5 nM hGH treatment. Thegrowth hormone antagonist (GHA) was used as a positive control.

FIG. 7. High throughput screening (HTS) platform for the identificationof GHR inhibitors. Mouse L cells expressing human GHR were transfectedwith SOCS2 reporter luciferase plasmids. Cells were serum starved,pre-treated with test compounds (numbered as shown) and then GH. Dataare normalized to CMV renilla luciferase and expressed as relative lightunits (RLU).

FIG. 8A. Mouse growth hormone receptor protein sequence. SEQ ID NO: 1 isdepicted: Residue 1-24 (italic), Signal peptide; Residue 25-264(underlined), GH binding protein; Residue 274-297 (bold), Transmembranedomain.

FIG. 8B. Structure analysis of mouse growth hormone receptor for epitopeselection. Epitope score was determined by a proprietary algorithm.

FIG. 9A. Complex of human growth hormone (GH) with its soluble bindingprotein (GHBP) (PDB-1HWG, visualized by PyMOL). A) Surface plot of the 5selected epitopes (human homologous regions).

FIG. 9B. Ribbon plot of the 5 selected epitopes. Dark Blue: GH; Green,GHBP; regions shaded in purple (C562), light brown (C570), red (C592),white (C576) and turquoise blue (C565) indicate the selected epitopesthat are targeted by monoclonal antibodies. Note, since only the humanGH-GHR complex crystal structure is available, high-lighted regions arethe human equivalent regions of selected mouse GHR epitopes.

FIG. 9C. Monoclonal anti-GHR antibody (C592) specifically recognizesmembrane-bound GHR (indicated by arrows).

FIG. 9D. High concentration of monoclonal anti-GHR antibody (C592)treatment leads to GHR clustering and endocytosis (indicated by arrows).

FIG. 10. Monoclonal antibodies and their target epitopes used in theexperiments of FIGS. 8 and 9.

FIG. 11A. Screening of antagonistic or agonistic monoclonal antibodiesagainst GHR. A). Mouse L cells were cultured to 80-90% confluency. Cellswere then switched to DMEM with low glucose (0.3 g/L) and low FBS (0.5%)for 24 hours. Cells were incubated with monoclonal antibodies (indicatedby serial numbers) for 1 hour, then treated with 5 nM Growth Hormone(GH) for 5 minutes, and assayed for STAT5 phosphorylation by WesternBlot. STAT5 phosphorylation levels were normalized to total STAT5 anddata are shown as percentage of no antibody treatment.

FIG. 11B. Downregulation of the GH induced phosphorylation of STAT5 bymonoclonal antibody C592.

FIG. 12A. Mice expressing a novel inhibitory growth hormone antibodydescribed in this application were protected against chemotherapy, 4week old CD1 male (M) and female (F) mice were immunized with human GHwith booster doses injected at week 10. A) The mice exhibited a loss inbody weight compared with controls indicating that they had generatedanti-human GH antibodies that cross-reacted with mouse GH and in turnslowed growth.

FIG. 12B. Slot-blot analysis of serum from immunized mice showedreactivity to human GH in 34 out of 40 mice confirming that antibodieshad been generated.

FIG. 12C. GH-immunized mice that were given a booster dose of human GHimmunization at 6 months and then treated with cyclophosphamide (CP) asshown were better able than controls (non-immunized) to regain bodyweight.

FIG. 12D. Complete blood counts also indicated that GH-immunized miceshowed better blood cell profile 7 days after cyclophosphamide (CP)treatment.

DETAILED DESCRIPTION

Reference will now be made in detail to presently preferredcompositions, embodiments, and methods of the present invention, whichconstitute the best modes of practicing the invention presently known tothe inventors. The Figures are not necessarily to scale. However, it isto be understood that the disclosed embodiments are merely exemplary ofthe invention that may be embodied in various and alternative forms.Therefore, specific details disclosed herein are not to be interpretedas limiting, but merely as a representative basis for any aspect of theinvention and/or as a representative basis for teaching one skilled inthe art to variously employ the present invention.

Except in the examples, or where otherwise expressly indicated, allnumerical quantities in this description indicating amounts of materialor conditions of reaction and/or use are to be understood as modified bythe word “about” in describing the broadest scope of the invention.Practice within the numerical limits stated is generally preferred.Also, unless expressly stated to the contrary: percent, “parts of,” andratio values are by weight; “R” groups include H, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₆₋₁₄ aryl (e.g., phenyl, halo, or C₄₋₁₄ heteroaryl; thedescription of a group or class of materials as suitable or preferredfor a given purpose in connection with the invention implies thatmixtures of any two or more of the members of the group or class areequally suitable or preferred; description of constituents in chemicalterms refers to the constituents at the time of addition to anycombination specified in the description, and does not necessarilypreclude chemical interactions among the constituents of a mixture oncemixed; the first definition of an acronym or other abbreviation appliesto all subsequent uses herein of the same abbreviation and appliesmutatis mutandis to normal grammatical variations of the initiallydefined abbreviation; and, unless expressly stated to the contrary,measurement of a property is determined by the same technique aspreviously or later referenced for the same property.

It is also to be understood that this invention is not limited to thespecific embodiments and methods described below, as specific componentsand/or conditions may, of course, vary. Furthermore, the terminologyused herein is used only for the purpose of describing particularembodiments of the present invention and is not intended to be limitingin any way.

It must also be noted that, as used in the specification and theappended claims, the singular form “a,” “an,” and “the” comprise pluralreferents unless the context clearly indicates otherwise. For example,reference to a component in the singular is intended to comprise aplurality of components.

Throughout this application, where publications are referenced, thedisclosures of these publications in their entireties are herebyincorporated by reference into this application to more fully describethe state of the art to which this invention pertains.

The term “subject” refers to a human or animal, including all mammalssuch as primates (particularly higher primates), sheep, dog, rodents(e.g., mouse or rat), guinea pig, goat, pig, cat, rabbit, and cow.

Abbreviation

AKT, V-Akt Murine Thymoma Viral Oncogene Homolog, Protein Kinase B.

B16Flu, Green fluorescent protein (GFP) expressing B16 mouse malignantmelanoma cells.

BM, Bone marrow.

cAMP, Cyclic adenosine monophosphate.

CMV, Cytomegalovirus immediate-early promoter.

CP, Cyclophosphamide.

df/df, Ames dwarf

DNA, Deoxyribonucleic Acid.

ERK, Extracellular signal-regulated kinases.

FBS, Fetal bovine serum.

GH, Growth hormone.

GHA, growth hormone antagonist.

GHR, Growth hormone Receptor.

GHR/BP, Growth hormone Receptor/growth hormone binding protein.

GHRD, Growth hormone Receptor deficiency.

GHRH, Growth hormone releasing hormone.

GHRKO, Growth hormone Receptor knockout.

HEK293, Human embryonic kidney cell 293.

hGH, Human growth hormone.

HSP72, Heat shock protein 72.

IGF-1, Insulin-like growth factor 1.

IGF-IR, IGF-1 receptor.

MEK, Mitogen-activated protein kinase kinase.

PB, Peripheral blood.

PBMC, Peripheral blood mononuclear cells.

Pit-1, POU domain, class 1, transcription factor 1.

PKA, Protein kinase A.

Prop-1, Prophet Of Pit1.

RAS, Rat sarcoma.

RLU, Relative light units.

S6K, Ribosomal Protein S6 Kinase.

Sch9, Serine/threonine-protein kinase SCH9.

SOCS, Suppressor of cytokine signaling.

STAT5, Signal Transducer and Activator of Transcription 5.

Tor, Target of rapamycin.

UV, Ultraviolet.

WBC, White blood cells.

WT, Wild type.

In various embodiments of the invention, compounds and methods foralleviating symptoms of various diseases, conditions, and treatments areprovided. In particular, these compounds and methods can be used totreat acromegaly, chemotherapy or other therapies involving toxins thatdamage normal cells, cancer, diabetes, immunodepression,immunosuppression, immunosenescence, immunodeficiency, Alzheimer's,aging, and diseases and conditions benefiting from cellular and tissueregeneration. Typically, the compounds and methods of the invention areused to treat ailments related to or caused by the expression (or overexpression) of GH, GHR, STAT5, IGF-1 and/or SOCS and of proteinsregulated by human GH, GHR, STAT5, SOCS, IGF-1, and insulin.

The present invention solves one or more problems of the prior art byproviding in at least one embodiment, a compound for treating diseasesor conditions by causing inhibition of the activity or expression of GH,GHR, STAT5, IGF-1 and/or SOCS and of proteins regulated by human GH,GHR, STAT5, SOCS, IGF-1, and insulin is provided. The compound of thisembodiment has formula I:

-   -   or a pharmaceutically acceptable salt thereof;        wherein:        R₁ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen, and        R₂ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen. In a        refinement, R₁ and R₂ are each independently hydrogen, methyl,        ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,        t-butyl, or phenyl.

In another embodiment, a compound that is useful for the treatment ofdiseases or conditions by causing inhibition of the activity orexpression of GH, GHR, STAT5, IGF-1 and/or SOCS and of proteinsregulated by human GH, GHR, STAT5, SOCS, IGF-1, and insulin is provided.The compound of this embodiment has formula II:

-   -   or a pharmaceutically acceptable salt thereof;        wherein;        R₁ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen, and        R₂ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen. In a        refinement, R₁ and R₂ are each independently hydrogen, methyl,        ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,        t-butyl, or phenyl.

In another embodiment, a compound that is useful for the treatment ofdiseases or conditions by causing inhibition of the activity or aexpression of GH, GHR, STAT5, IGF-1 and/or SOCS and of proteinsregulated by human GH, GHR, STAT5, SOCS, IGF-1, and insulin is provided.The compound of this embodiment has formula III:

-   -   or a pharmaceutically acceptable salt thereof;        wherein:        R₁ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₄ alkyl, or halogen, and        R₂ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen. In a        refinement, R₁ and R₂ are each independently hydrogen, methyl,        ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,        t-butyl, or phenyl.

In another embodiment, a compound that is useful for the treatment ofdiseases or conditions by causing inhibition of the activity orexpression of GH, GHR, STAT5, IGF-1 and/or SOCS and/or of proteinsregulated by human GH, GHR, STAT5, SOCS, IGF-1, and insulin is provided.The compound of this embodiment has formula IV:

-   -   or a pharmaceutically acceptable salt thereof;        wherein:        R₁ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen, and        R₂ is hydrogen, NO₂, SO₃H, NH₃, C₁₋₈ alkyl, or halogen. In a        refinement, R₁ and R₂ are each independently hydrogen, methyl,        ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,        t-butyl, or phenyl.

In still another embodiment, the compounds set forth above in formulaeI-IV are uses to treating diseases or conditions selected from the groupconsisting of acromegaly, cancer, diabetes, Alzheimer's, and aging. Thecompounds and inhibition of the pathways by the compounds are alsouseful to protect from chemotherapy toxicity and promote multi-systemstem cell based regeneration (C. W. Cheng et al. Cell Stem Cell, 2014;14 (6); 810 DOI: 10.1016/j.stem.2014.04.014). In this regard, a methodfor treating diseases or conditions with alterations in signaling genesrelated to human GH, GHR, STAT5, and SOCS is provided. In this context,the term “related to” means that the signaling genes are part or asignaling pathway that leads to expression of GH, GHR, STAT5, and SOCS.This includes a step of identifying a subject having a disease orcondition in which signaling genes or the proteins which they encoderelated to human GH, GHR, STAT5, and SOCS or contribute to the diseaseor condition. A therapeutically effective amount of a compound selectedfrom the group consisting of compounds having formulae I-IV andcombinations thereof is administered to the subject.

The compounds set forth above are administered to the subject in atherapeutically effective amount so that symptoms of acromegaly, cancer,diabetes, Alzheimer's, or aging are alleviated. In a refinement, suchamounts will generally be from about 0.1 to about 300 mg per kg ofsubject body weight depending on the specific compound being used and onits effects on the activity or expression of GH, GHR, STAT5, SOCS, IGF-1and insulin. Typical doses are from about 1 to about 5000 mg per day foran adult subject of normal weight.

The compounds of the present invention may form pharmaceuticallyacceptable salts with both organic and inorganic acids or bases. Forexample, the acid addition salts of the basic compounds are preparedeither by dissolving the free base in aqueous or aqueous alcoholsolution or other suitable solvents containing the appropriate acid andisolating the salt by evaporating the solution. Examples ofpharmaceutically acceptable salts are hydrochlorides, hydrobromides,hydrosulfates, etc. as well as sodium, potassium, and magnesium, etc.salts. The compounds having formula I-IV can contain one or severalasymmetric carbon atoms. The invention includes the individualdiastereomers or enantiomers, and the mixtures thereof. The individualdiastereomers or enantiomers may be prepared or isolated by methodsalready well-known in the art.

Pharmaceutical compositions include the compounds set forth above or asalt therefore and a pharmaceutical carrier. Typically thepharmaceutical compositions am divided into dosage units. Examples ofdosage unit forms include, but are not limited to, pills, powders,tablets, capsules, aqueous and nonaqueous oral solutions andsuspensions, and parenteral solutions.

Examples of suitable pharmaceutical carriers include, but are notlimited to, water, sugars (e.g., lactose and sucrose), starches (e.g.,corn starch and potato starch), cellulose derivatives (e.g., sodiumcarboxymethyl cellulose, and methyl cellulose), gelatin, talc, stearicacid, magnesium stearate, vegetable oils (e.g., peanut oil, cottonseedoil, sesame oil, olive oil, etc.), propylene glycol, glycerin, sorbitol,polyethylene glycol, water, agar, alginic acid, saline, and otherpharmaceutically acceptable materials.

The percentage of the active ingredients in the pharmaceuticalcompositions can be varied within wide limits. In one refinement, aconcentration of at least 10% in a solid composition and at least 2% ina primary liquid composition is used. The most useful compositions havea higher percentage of the active which a much higher proportion of theactive ingredient is present.

Routes of administration of the subject compound or its salts are oralor parenteral. For example, a useful intravenous dose is between 1 and50 mg and a useful oral dosage is between 5 and 800 mg.

In still another embodiment, a method for treating diseases related toor caused by growth hormone activity is provided. A subject having adisease or condition caused by the activity of growth hormone or genesregulated by it including GHR, IGF-1 and insulin is identified. Atherapeutically effective amount of antibodies that target the growthhormone receptor and/or the growth hormone is administered to thesubject. In this embodiment, inhibitory anti-growth hormone (anti-GH) oranti growth hormone receptor (anti-GHR) monoclonal antibodies are usedto reduce growth hormone (GH) action and consequently the Insulin GrowthFactor 1 (IGF-1) and insulin activity. Because of the pro-growth effectof GH and IGF-1 on certain tumor cells and because GH/IGF-1 signalingplays an important role in mammalian aging, this invention has a greatpotential to provide an antibody-based drug to: a) attenuate/delay thegrowth of GH- and/or IGF-1 dependent tumor cells; b) induceorganism-level protection against acute stress, e.g. chemotherapy- orcancer therapy-associated toxicity to normal tissue, radiation inducedcellular toxicity, or other toxic drugs and compounds; c) enhance thetherapeutic index of existing chemotherapy and other cancer therapies;d) modulate risk factors associated with age-related diseases; e)attenuate/delay changes in biomarkers associated with aging; f) promotemulti-system stem cell-based regeneration; g) reduce or delay theincidence of Alzheimer and retard its progression.

The following examples illustrate the various embodiments of the presentinvention. Those skilled in the art will recognize many variations thatare within the spirit of the present invention and scope of the claims.

Assay procedures: The following assays are used to screen foractivity: 1. STAT5 phosphorylation assay and 2. Luciferase reporterassays.

The JAK/STAT signaling pathway is the principal signaling mechanism fora variety of growth factors and cytokines. In the growth hormonesignaling pathway, dimerization of the GHR in response to GH bindingleads to the activation of JAK by transphosphorylation. Activated JAKphosphorylates STAT5, which can then enter the nucleus and activatetranscription of target genes. Inhibition of STAT5 phosphorylation willbe used to analyze GHR antagonistic properties of the test compounds.

Mouse L fibroblasts engineered to express mouse GHR were obtained fromDr. John Kopchick. Serum starved L cells (0.5% FBS for 24 hours) werepre-treated for 30 minutes with 10 μM of each compound identified as ahit from the luciferase assays. Following this, cells were treated with5 or 10 nM hGH for 10 minutes. Cells were collected and processed forwestern blotting with phospho-STAT5 and total STAT5 antibodies (CellSignaling). The GHR antagonist, G120K is used as a positive control.Band intensity was analyzed using ImageJ and STAT5 phosphorylationnormalized to total STAT5 levels. Compounds that inhibited STAT5phosphorylation were selected for further drug optimization.

In order to perform high throughput screening (HTS) for inhibitors ofGHR, HEK293 cell lines permanently transfected were established with aluciferase reporter driven by either c-fos or SOCS2 promoter (FIG. 7).

The SOCS (Suppressor of Cytokine Signaling) proteins are induced by G1and act as negative regulators of cytokine signaling pathways [30, 31].Expression of the proto-oncogene c-fos is also induced by GH via theRas/MEK/ERK pathway [32]. Luciferase reporter constructs where theluciferase gene is under the control of promoters of either SOCS2 orc-fos have been stably transfected into human embryonic kidney, HEK293cells. Cells were serum starved (0.5% FBS) for 24 hours to preventinterference by other growth factors and then treated with 5 or 10 nMhGH (established in dose response studies) and 10 μM of each testcompound for 24 hours. This is followed by lysis and analysis ofluciferase activity using the luciferase reporter assay (Promega). Eachcompound is tested in triplicate in a 96 well plate format.

Three potential lead compounds were identified that inhibit GH-GHRsignaling (DSR 34, 37 and 38) (FIGS. 5 A and B) using our primaryscreening process. FIG. 5 shows luciferase reporter assays for the threecompounds, DSR 34, 37 and 38. All three compounds inhibited GH inducedincrease in c-fos and SOCS2 reporter activity (FIGS. 5 A and B).Further, the compounds could inhibit reporter activities to below thatof untreated controls, c-fos reporter activity is 57%, 27% and 46.5% ofuntreated controls whereas SOCS2 reporter activity is 76%, 61% and 57%of untreated controls for compounds 34, 37 and 38 respectively. Thesecompounds were also tested for their ability to inhibit GH inducedphosphorylation of STAT5 in mouse L cells expressing human GHR (FIG. 6).All three compounds are able to inhibit phosphorylation of STAT5 (FIG.6). These compounds were further optimized and generated an additionallead (compound 22) as shown below (FIG. 7).

The structure of compounds 22, 34, 37, and 38 are as follows:

-   -   or pharmaceutically acceptable salts thereof;

To test whether a reduction in GH/IGF-1 signaling by inhibiting GH usinganti GH antibodies would also protect mice from chemotherapy toxicity,antibodies against mouse Growth Hormone were generated. Four (4) weekold CD1 male and female mice were immunized with human GH with boosterdoses injected at 10 weeks. The mice exhibit a loss in body weightcompared with controls indicating that they had generated anti-human GHantibodies that cross-reacted with mouse GH and in turn slowed growth(FIG. 12A). Slot-blot analysis of serum from immunized mice showedreactivity with hGH in 34/40 mice confirming that antibodies weregenerated (FIG. 12B). Mice that were given a booster dose of hGH at 6months and then treated with cyclophosphamide (CP) as shown were betterable than controls to regain body weight (FIG. 12C). Complete bloodcounts also indicated that these mice were more resistant to CP toxicity(FIG. 12D). STI indicates short-term immunization.

Monoclonal antibodies against various regions of the mouse GHRextracellular domain were developed. Growth hormone binds asymmetricallyto the growth hormone receptor homodimer, leading to a rotation of theGHR subunits relative to each other. The current understanding of GH-GHRbinding suggests that this receptor dimer conformation change ispropagated through the transmembrane domain and lead to signaltransduction to downstream tyrosine kinase. Depending on the epitopelocation and binding affinity, the binding of monoclonal antibodies canlead to receptor conformation change, aggregation, and protein-proteininteraction alteration that result in either stimulatory or inhibitoryeffects on GHR (see FIG. 11). For example, monoclonal antibody C562targets the N-terminal region of the GHR (FIGS. 9 and 10, targetpeptide—SEQ ID NO 2: TEGDNPDLKTPG, lavender shaded), with is approximateto the GH binding site and thus interferes ligand-receptor interaction;monoclonal antibody C565 binds GHR in a region (FIGS. 9 and 10, targetpeptide—SEQ ID NO 3: ESKWKVMGPIWL, aqua blue shaded) that may interfereboth the dimerization of GHR subunits and the re-orientation of thesubunits after GH binding; monoclonal antibody C592 targets the GHRregion (FIGS. 9 and 10, target peptide—SEQ ID NO 6: TVDEIVQPDPPI, redshade) that sits in a “hinge” of the GHR extracellular domain. C592binding may interfere GH binding and promote GHR clustering, endocytosisand degradation (FIGS. 9C and D). These antagonistic and agonisticmonoclonal antibodies and their derivatives (e.g. Fab, F(ab′)₂preparations) could be potent therapeutic agents in modulating GH-GHRaction in vivo. Therefore, novel and effective antibodies binding to theGHR to affect its activity have been described.

While exemplary embodiments are described above, it is not intended thatthese embodiments describe all possible forms of the invention. Rather,the words used in the specification are words of description rather thanlimitation, and it is understood that various changes may be madewithout departing from the spirit and scope of the invention.Additionally, the features of various implementing embodiments may becombined to form further embodiments of the invention.

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1.-15. (canceled)
 16. A method for treating diseases or conditionscaused by growth hormone activity, the method comprising: identifying asubject having a disease or condition caused by the activity of growthhormone or genes regulated by it including GHR, IGF-1 and insulin; andadministering a therapeutically effective amount of antibodies thattarget the growth hormone receptor and/or the growth hormone.
 17. Themethod of claim 16 wherein the antibodies are monoclonal antibodies. 18.The method of claim 16 wherein the diseases or conditions are selectedfrom the group consisting of acromegaly, chemotherapy or other therapiesinvolving toxins that damage normal cells, cancer, diabetes,immunodepression, immunosuppression, immunosenescence, immunodeficiency,Alzheimer's disease, aging, and diseases and conditions benefiting fromcellular and tissue regeneration.